Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes.
Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes.
复制标题
傅立叶转化离子回旋共振质谱法作为表征多聚体膜蛋白复合物的平台。
DOI：
10.1007/s13361-017-1799-4
复制
发表时间：
2018-01
影响因子：
3.2
通讯作者：
Campuzano IDG
中科院分区：
文献类型：
--
作者：
Lippens JL;Nshanian M;Spahr C;Egea PF;Loo JA;Campuzano IDG
膜蛋白的特性一直受到表达、纯化和增溶挑战的阻碍。在用于表征它们的几种生物物理技术中，天然质谱学(MS)已经成为分析膜蛋白和复合体的有力工具。本文利用FT-ICR和QToF两个MS平台，在非变性条件下对水通道蛋白AqpZ(AquaporinZ)进行了分析。在两种MS平台上，在各种仪器条件下，这种97 kDa的膜蛋白复合体可以很容易地从辛基糖苷(OG)洗涤剂胶束中释放出来。增加FT-ICR碰撞池的碰撞能，可以不同程度地从OG胶束中释放四聚体(97 KDa)，并解离为三聚(72 KDa)和单体(24 KDa)取代基。Q-ToF上的串联MS产生了更高强度的四聚体信号，并且，根据所选择的m/z区域，观察到的单体信号的强度有所不同。选择高于预期蛋白质信号分布的m/z范围的前体离子，然后进行温和的碰撞活化，能够有效地释放具有高S/N比的AqpZ。在两种仪器上获得的四聚体电荷态分布表明，由于不同程度的N-末端甲酰化，多种蛋白质形式重叠。
Membrane protein characterization is consistently hampered by challenges with expression, purification and solubilization. Among several biophysical techniques employed for their characterization, native-mass spectrometry (MS) has emerged as a powerful tool for the analysis of membrane proteins and complexes. Here, two MS platforms, the FT-ICR and QToF, have been explored to analyze the homotetrameric water channel protein, AquaporinZ (AqpZ) under non-denaturing conditions. This 97 kDa membrane protein complex can be readily liberated from the octylglucoside (OG) detergent micelle under a range of instrument conditions on both MS platforms. Increasing the applied collision energy of the FT-ICR collision cell yielded varying degrees of tetramer (97 kDa) liberation from the OG micelles, as well as dissociation into the trimeric (72 kDa) and monomeric (24 kDa) substituents. Tandem-MS on the Q-ToF yielded higher intensity tetramer signal and, depending on the m/z region selected, the observed monomer signal varied in intensity. Precursor ion selection of an m/z range above the expected protein signal distribution, followed by mild collisional activation is able to efficiently liberate AqpZ with a high S/N ratio. The tetrameric charge state distribution obtained on both instruments demonstrated superpositioning of multiple proteoforms due to varying degrees of N-terminal formylation.
登录
查看更多内容
影响因子：
7.4
作者：
Calabrese AN;Watkinson TG;Henderson PJ;Radford SE;Ashcroft AE
通讯作者：
Ashcroft AE
完整膜蛋白复合物的质谱法。
影响因子：
14.8
作者：
通讯作者：
--
DOI：
10.1113/jphysiol.2014.283150
发表时间：
2015-01-15
期刊：
The Journal of physiology
影响因子：
--
作者：
Landreh M;Robinson CV
通讯作者：
Robinson CV
影响因子：
1.8
作者：
Lange, Oliver;Damoc, Eugen;Makarov, Alexander
通讯作者：
Makarov, Alexander
影响因子：
2.9
作者：
le Coutre, J;Whitelegge, JP;Faull, KF
通讯作者：
Faull, KF